Pharmaceutical compositions containing beta-lapachone, or derivatives or analogs thereof, and methods of using same

ABSTRACT

Beta-lapachone, which is poorly soluble in most pharmaceutically acceptable solvents, has demonstrated significant antineoplastic activity against human cancer lines. The present invention overcomes this significant limitation by teaching novel pharmaceutical compositions comprising a therapeutically effective amount of Beta-lapachone, or a derivative or analog thereof, and a pharmaceutically acceptable solubilizing carrier molecule, which may be at water-solubilizing carrier molecule such as hydroxypropyl-β-cyclodextrin, or an oil-based solubilizing carrier molecule, for enhancing the solubility of Beta-lapachone in aqueous solution. The therapeutically effective amount of Beta-lapachone, or a derivative or analog thereof, may be complexed with the pharmaceutically acceptable solubilizing carrier molecule in aqueous solution. The novel pharmaceutical compositions may be administered with a second anticancer agent or in combination with radiation therapy. A formulation of Beta-lapachone or a derivative or analog thereof, complexed with a pharmaceutically acceptable solubilizing carrier molecule, wherein the complex can be freeze-dried and when subsequently reconstituted in aqueous solution is substantially soluble is also disclosed. Emulsions of Beta-Lapachone in a pharmaceutically acceptable fat emulsion vehicle are also provided. Also disclosed are methods for treating cancer by administering to a patient the novel pharmaceutical compositions and formulations. Pharmaceutical kits are also provided.

RELATED APPLICATION

This application is a National Phase Application which claims priorityfrom PCT/US02/24262, filed Jul. 31, 2002; which is acontinuation-in-part of and claims priority from U.S. Ser. No.09/975,776, filed Oct. 10, 2001; now U.S. Pat. No. 6,962,944 whichclaims priority from U.S. Ser. No. 60/308,935 filed Jul. 31, 2001, eachof which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical compositions andformulations, as well as methods of administering these pharmaceuticalcompositions and formulations, which comprise β-lapachone(Beta-lapachone), or a derivative or analog thereof, complexed orcombined with a solubilizing carrier molecule for enhancing thesolubility of β-lapachone in different solvent systems.

BACKGROUND OF THE INVENTION

Over 1.22 million new cancer cases will be diagnosed in the U.S. in theyear 2001 alone. With more than 563,000 deaths annually, cancer is thesecond leading cause of death behind heart disease (UBS Warburg “DiseaseDynamics: The Cancer Market”, Nov. 8, 2000). Surgery and radiotherapymay be curative if the disease is found early, but current drugtherapies for metastatic disease are mostly palliative and seldom offera long-term cure. Even with the new chemotherapies entering the market,improvement in patient survival is measured in months rather than inyears, and the need continues for new drugs effective both incombination with existing agents as first line therapy and as second andthird line therapies in treatment of resistant tumors.

In the past, the most successful drug treatment regimens have combinedtwo or more agents, each of which has a different mechanism of actionand each of which has antitumor activity when used individually. Eventhough their mechanisms of action differ, most of the agents currentlyused for chemotherapy of cancer, including alkylating agents, platinumanalogs, anthracyclines and the camptothecin family of topoisomeraseinhibitors, have in common the property of severely damaging DNA, hencetheir designation as “DNA-damaging agents”. Radiotherapy workssimilarly. Most DNA-damaging agents as well as the microtubule-targetingagents (e.g., paclitaxel) cause the arrest of cells at the G₂/Mtransition phase of the cell cycle, a major cell cycle checkpoint wherecells make a commitment to repair DNA or to undergo apoptosis if DNAdamage al., Molecular and Biochemical Parasitology 1:167–176 (1998)(substituents at the 2- and 3-positions)).

As a single agent, β-lapachone has demonstrated significantantineoplastic activity against human cancer cell lines atconcentrations typically in the range of 1-10 μM (IC₅₀). Cytotoxicityhas been demonstrated in transformed cell lines derived from patientswith promyelocytic leukemia (Planchon et al., Cancer Res., 55 (1996)3706), prostate (Li, C. J., et al., Cancer Res., 55 (1995) 3712),malignant glioma (Weller, M. et al., Int. J. Cancer, 73 (1997) 707),hepatoma (Lai, C. C., et al., Histol Histopathol, 13 (1998) 8), colon(Huang, L., et al., Mol Med, 5, (1999) 711), breast (Wuertzberger, S.M., et al., Cancer Res., 58 (1998) 1876), ovarian (Li, C. J. et al.,Proc. Natl. Acad. Sci. USA, 96(23) (1999) 13369-74), pancreatic (Li, Y.,et al., Mol Med, 6 (2000) 1008; Li, Y. Z., Mol Med, 5 (1999) 232), andmultiple myeloma cell lines, including drug-resistant lines (Li, Y., MolMed, 6 (2000) 1008). No cytotoxic effects were observed on normal freshor proliferating human PBMC (Li, Y., Mol Med, 6 (2000) 1008).

β-lapachone has been shown to be a DNA repair inhibitor that sensitizescells to DNA-damaging agents including radiation (Boothman, D. A. etal., Cancer Res, 47 (1987) 5361; Boorstein, R. J., et al., Biochem.Biophys. Commun., 117 (1983) 30). Although its exact intracellulartarget(s) and mechanism of cell killing remain unknown, β-lapachone hasalso shown potent in vitro inhibition of human DNA Topoisomerases I (Li,C. J. et al., J. Biol. Chem., 268 (1993) 22463) and II (Frydman, B. etal., Cancer Res. 57 (1997) 620) with novel mechanisms of action. Unliketopoisomerase “poisons” (e.g., camptothecin, etoposide, doxorubicin)which stabilize the covalent topoisomerase-DNA complex and inducetopoisomerase-mediated DNA cleavage, β-lapachone interacts directly withthe enzyme to inhibit catalysis and block the formation of cleavablecomplex (Li, C. J. et al., J. Biol. Chem., 268 (1993) 22463) or with thecomplex itself, causing religation of DNA breaks and dissociation of theenzyme from DNA (Krishnan, P. et al., Biochem Pharm, 60 (2000) 1367).β-lapachone and its derivatives have also been synthesized and tested asanti-viral and anti-parasitic agents (Goncalves, A. M., et al., Mol.Biochem. Parasitology, 1 (1980) 167–176; Schaffner-Sabba, K., et al., J.Med Chem., 27 (1984) 990–994).

More specifically, β-lapachone appears to work by disrupting DNAreplication, causing cell-cycle delays in G1 and/or S phase, inducingeither apoptotic or necrotic cell death in a wide variety of humancarcinoma cell lines without DNA damage and independent of p53 status(Li, Y. Z. et al (1999); Huang, L. et al.). Topoisomerase I is an enzymethat unwinds the DNA that makes up the chromosomes. The chromosomes mustbe unwound in order for the cell to use the genetic information tosynthesize proteins; β-lapachone keeps the chromosomes wound tight, sothat the cell cannot make proteins. As a result, the cell stops growing.Because cancer cells are constantly replicating and circumvent manymechanisms that restrict replication in normal cells, they are morevulnerable to topoisomerase inhibition than are normal cells.

Another possible intracellular target for β-lapachone in tumor cells isthe enzyme NAP(P)H:quinone oxidoreductase (NQO1). Biochemical studiessuggest that reduction of β-lapachone by NQO1 leads to a “futilecycling” between the quinone and hydroquinone forms with a concomitantloss of reduced NADH or NAD(P)H (Pink, J. J. et al., J. Biol Chem., 275(2000) 5416). The exhaustion of these reduced enzyme cofactors may be acritical factor for the activation of the apoptotic pathway afterβ-lapachone treatment.

As a result of these findings, β-lapachone is actively being developedfor the treatment of cancer and tumors. In WO00/61142, for example,there is disclosed a method and composition for the treatment of cancer,which comprises the administration of an effective amount of a firstcompound, a G1 or S phase drug, such as a β-lapachone, in combinationwith a G2/M drug, such as a taxane derivative. Additionally, U.S. Pat.No. 6,245,807 discloses the use of β-lapachone, amongst otherβ-lapachone derivatives, for use in the treatment of human prostatedisease.

One obstacle, however, to the development of pharmaceutical formulationscomprising β-lapachone for parenteral and topical administration is thelow solubility of β-lapachone in pharmaceutically acceptable solvents.β-lapachone is highly insoluble in water and has only limited solubilityin common solvent systems used for topical and parenteraladministration, specifically for intravenous and cutaneous delivery ofdrugs. As a result, there is a need for improved formulations ofβ-lapachone for parenteral and topical administration, which are bothsafe and readily bioavailable to the subject to which the formulation isadministered.

SUMMARY OF THE INVENTION

The present invention is directed generally to pharmaceuticalcompositions containing β-lapachone for use in the treatment ofmammalian cancers and which overcome the disadvantages and obstacles ofprior art compositions. More specifically, the invention is directed topharmaceutical compositions containing β-lapachone, or a derivative oranalog thereof, and a pharmaceutically acceptable solubilizing carriermolecule for use in the treatment of mammalian cancers, including lung,breast, colon, ovarian and prostate cancers, multiple myeloma, malignantmelanoma, non-melanoma skin cancers, as well as proliferation disordersand dermatological conditions such as psoriasis. The pharmaceuticalcomposition may be complexed or combined with the pharmaceuticallyacceptable solubilizing carrier molecule to form a unitary compositionor an inclusion complex. The pharmaceutically acceptable solubilizingcarrier molecule is advantageously a water-solubilizing carrier moleculeor an oil-based solubilizing carrier molecule.

The present invention provides pharmaceutical compositions ofβ-lapachone, or a derivative or analog thereof, and a pharmaceuticallyacceptable solubilizing carrier molecule that enhances the solubility ofthe β-lapachone and renders it bioavailable in mammalian bodies andsuitable for parenteral and topical administration. The concentration ofβ-lapachone in solution is preferably at least 1 mg/ml, more preferablyat least 3 mg/ml, even more preferably at least 5 mg/ml. Forconcentrated pharmaceutical compositions, we contemplate concentrationsof β-lapachone of 10 mg/ml or greater.

The present invention also provides pharmaceutical compositionscontaining β-lapachone and pharmaceutically acceptable solubilizingcarrier molecules in combination with a taxane derivative or otheranticancer agent, for use in the treatment of mammalian cancers.

The present invention also provides formulations of β-lapachone, or aderivative or analog thereof, complexed with pharmaceutically acceptablesolubilizing carrier molecules, wherein the complex can be freeze-driedand when subsequently reconstituted in aqueous solution is substantiallysoluble.

The present invention further provides methods for treating mammaliancancers by administering to a patient the pharmaceutical compositionsand formulations of the present invention.

The present invention further provides methods for treating cancer andfor treating dermatologic conditions by administering to a patientafflicted with cancer or a dermatologic condition, an analog orderivative of β-lapachone, such as 4-aceotoxy-β-lapachone or4-acetoxy-3-bromo-β-lapachone.

The present invention also provides pharmaceutical kits which compriseone or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of β-lapachone, or aderivative or analog thereof. Such kits may include, if desired, one ormore of various conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, etc. Printed instructions, either asinserts or as labels, indicating quantities of the components to beadministered, guidelines for administration, and/or guidelines formixing the components, may also be included in the kit.

The above description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be understood, and in order that the presentcontributions to the art may be better appreciated. Other objects andfeatures of the present invention will become apparent from thefollowing detailed description considered in conjunction with theaccompanying drawings. It is to be understood, however, that thedrawings are designed solely for the purposes of illustration and not asa definition of the limits of the invention, for which reference shouldbe made to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the appendedfigures in which:

FIG. 1 is a bar graph illustrating the relative solubility ofβ-lapachone in aqueous solutions of various solubilizing agents;

FIG. 2 is a bar graph illustrating the solubility of β-lapachone as afunction of hydroxypropyl-β-cyclodextrin concentration (HPBCD);

FIG. 3 is an HPLC chromatogram of a 5 mg/ml β-lapachone solution in 20%hydroxypropyl-β-cyclodextrin concentration;

FIG. 4 is a chart illustrating the inhibition of cancer cell survival byβ-lapachone and Taxol®;

FIG. 5 is a chart showing the growth inhibitory profile of β-lapachonein combination with Taxol® against ovarian tumor cell lines asdetermined by MTT assay;

FIG. 6 is an isobologram showing synergistic drug-drug interaction forβ-lapachone and Taxol® in the OVCAR-3 ovarian tumor cell line;

FIG. 7 is an isobologram showing synergistic drug-drug interaction forβ-lapachone and Taxol® in the MDAH-2774 ovarian tumor cell line;

FIG. 8 is a graph illustrating the cytotoxic effect of β-lapachone onCisplatin-sensitive (A2780s) and Cisplatin-resistant (A2780DDP) ovariancancer lines;

FIG. 9 is a bar graph illustrating the synergistic effect of β-lapachoneplus Taxol® in mouse model of human ovarian carcinoma;

FIG. 10 is a bar graph illustrating that β-lapachone is equallyefficacious in the mouse model of human ovarian carcinoma whenformulated in HPBCD solution;

FIG. 11 illustrates anti-tumor activity of β-lapachone and Taxol® inhuman breast cancer xenograft model;

FIG. 12 illustrates preferred β-lapachone analogs and derivatives inaccordance with the present invention;

FIG. 13 is a schematic illustrating the injection of RPMI 8226 MM cellsand the resulting tumor formation in Bg-Nu-Xid mice;

FIG. 14 is a graph showing the average weight pattern of mice during thestudy;

FIG. 15 is a graph showing the effect on β-lapachone/Hydroxypropylβ-cyclodextrin on tumor volume;

FIG. 16 is a graph showing the effect of β-lapachone/hydroxypropylβ-cyclodextrin on the survival of mice in both groups; and

FIG. 17 illustrates representative photomicrographs showing the effectof β-lapachone/hydroxypropyl β-cyclodextrin on tumors, as well as liver,spleen, lung, heart, brain and kidneys.

DETAILED DESCRIPTION OF THE INVENTION

β-lapachone, as well as its derivatives and analogs thereof (alsoreferred to herein as the “active compounds”), are described in Li, C.J. et al., J. Biol. Chem., 1993. These active compounds can beincorporated into pharmaceutical compositions suitable for parenteraladministration. Such compositions typically comprise the active compoundand a pharmaceutically acceptable carrier, excipient, diluent oradjuvant. However, the low solubility of β-lapachone in mostpharmaceutically acceptable solvents has been an obstacle to thedevelopment of a suitable formulation for parenteral and topicaladministration, particularly intravenous and cutaneous administration,respectively. Table 1 illustrates the limited solubility of β-lapachonein common solvent systems used for intravenous delivery of drugs.Preclinical pharmacokinetic data produced to date suggest that the idealpeak plasma concentration is in the range of 10 μg/ml. To achieve thisplasma concentration, an intravenous formulation must have a β-lapachoneconcentration approaching 10 mg/ml and be able to be diluted 5–×10× withsterile fluids for intravenous administration, such as saline or D5W.

TABLE 1 β-lapachone Solubility (mg/ml) Undiluted 5X dilution* SolventSystem (mg/ml) (mg/ml) Poloxamer 20% 2.0350 0.0331 Povidone K17 20%1.8250 0.0312 Povidone K12 20% 1.8600 0.0313 Tween 80 11.1700 1.6550EtOH 76% 10.6600 0.1025 PEG 400 11.6800 0.1400 Propylene Glycol 8.78000.0950 Trappsol 20% 1.4650 0.0300 *Diluted in 0.9% saline

The maximum solubility of β-lapachone in the solvents listed in Table 1was about 12 mg/ml. Upon dilution, the solubility decreased more thanthe dilution factor in all the systems. Although various preclinicalstudies have used a variety of common solvent systems, such as lipiodol,peanut oil, Cremophor/ethanol or PEG4000, for i.p. and i.v. dosing, noneof these approaches have yet demonstrated suitability for development ofan i.v. formulation for use in the clinic. Combining, mixing and/orcomplexing β-lapachone, its derivatives or analogs, with apharmaceutically-acceptable water-solubilizing carrier molecule, whichis advantageously hydroxypropyl-β-cyclodextrin (HPBCD) increases theaqueous solubility of β-lapachone with concentrations as high as 20mg/ml in 50% HPBCD solution as illustrated in Table 2.

TABLE 2 β-lapachone HPBCD in (highest conc.) Water (mg/ml) 10% 3.07 20%7.04 30% 10.78 40% 15.77 50% 19.74These β-lapachone/HPBCD solutions are stable for extended periods atroom temperature and can be further diluted with sterile fluids for IVadministration (e.g., sterile saline, D5W) and held for at least 24hours without precipitation of β-lapachone. The β-lapachone/HPBCDsolutions may also be sterile filtered, lyophilized and readilyreconstituted in water. Experimentation has determined thatHPBCD@20%/β-lapachone@5 mg/ml provides an excellent concentration foreasy lyophilization and relatively fast reconstitution. The invention isnot limited in this respect, however, and concentrations of β-lapachoneas low as 1 mg/ml have been prepared and determined to be stable andcapable of being lyophilized and reconstituted. The combining orcomplexation of β-lapachone with HPBCD also appears to improve thestability of β-lapachone to photoreduction compared with complexation ofβ-lapachone with ethanol solutions.

Further study of β-lapachone in aqueous HPBCD solutions has demonstratedthat the solubility of β-lapachone increases linearly with the increasein HPBCD concentration. Upon 10 to 100 times dilution, the decrease ofβ-lapachone concentration in all HPCD systems is proportional to thedilutions made.

Cyclodextrins are crystalline, nonhygroscopic cyclic oligomers ofα-D-glucopyranose derived from starch. As a result of a lack of rotationabout the bonds connecting the glucopyranose units, the cyclodextrinsare not cylindrical, but toroidal in shape. Because of this restrictedrotation they have a rigid structure with a central cavity whose sizevaries according to the number of glucopyranose units in the molecule.The three most common cyclodextins are α-cyclodextrin, β-cyclodextrinand γ-cyclodextrin, and which consist of six, seven and eightglucopyranose units respectively. Due to the arrangement of hydroxylgroups within the cyclodextrin molecule and the shape of the molecule,the internal surface of the cavity is hydrophobic, while the outsidesurface is hydrophilic. The primary hydroxyl groups are located on thenarrower (inner) side of the toroidal molecule, while the secondaryhydroxyl groups are located on the wider (outer) edge. This arrangementpermits the cyclodextrins to accommodate a wide variety of smallhydrophobic molecules within the hydrophobic cavity by forming aninclusion complex.

The HPBCD has seven glucopyranose units and has hydroxypropyl groupsattached to each glucopyranose unit on the outer surface of the toroidalstructure. The solubility of HPBCD in water has been shown to be farsuperior than that of β-cyclodextrin. The introduction of thehydroxypropyl groups into the β-cyclodextrin renders it more soluble bydisrupting the intramolecular hydrogen bonding between hydroxyl moietieson the cyclodextrin cavity. As a result, inclusion complexes formed byHPBCD will also have higher solubility in water compared to inclusioncomplexes formed by β-cyclodextrins. The degree of substitutiondetermines the solubility and complexation patterns. The lesser thesubstitution, the more the binding will be similar to that ofunsubstituted cyclodextrin in terms of binding, as well as solubility.Higher substitution renders the cyclodextrin more soluble in water butless binding. The degree of substitution of cyclodextrins is easilycontrolled.

When complexing β-lapachone, its derivatives or analogs, with awater-solubilizing carrier molecule in accordance with the presentinvention, the complexed solution generally becomes a unitarycomposition, or in the case where the water-solubilizing carriermolecule is a HPBCD, an inclusion complex is formed wherein theinsoluble β-lapachone, its derivatives or analogs, is within thecyclodextrin cavity. The invention is not limited, however, to theformation of a complex.

Although HPBCD is the preferred solubilizing agent, the invention is notlimited in this respect, and other water-solubilizing agents forcombining with β-lapachone, its derivatives or analogs, such asPoloxamer, Povidone K17, Povidone K12, Tween 80, ethanol,Cremophor/ethanol, polyethylene glycol 400, propylene glycol andTrappsol, are contemplated. Furthermore, the invention is not limited towater-solubilizing agents, and oil-based solubilizing agents such aslipiodol and peanut oil, may also be used.

Surfactants are also contemplated as part of the present invention forsolubilization of β-lapachone, its derivatives or analogs. It isnecessary, however that the surfactant(s) used must be present at a highenough level when β-lapachone, its derivatives or analogs, is diluted inwater so that there is sufficient surfactant to retain the β-lapachone,derivative or analog in solution. However, there cannot be too muchsurfactant to cause intolerable side effects.

Emulsions of β-lapachone, its derivatives or analogs, may also be formedand are contemplated by the present invention. Emulsions may be preparedwhich comprise a therapeutically effective amount of β-lapachone, itsderivatives or analogs, in one or more emulsifiers or emulsifying agentswhich may result in an oil-in-water-type emulsion for parenteraladministration. Suitable emulsifiers or emulsifying agents may include,but are not limited to, any pharmaceutically acceptable emulsifier,preferably phospholipids extracted from egg yolk or soy bean, syntheticphosphatidyl cholines or purified phosphatidyl cholines from vegetableorigin. Hydrogenated derivatives can also be used, such as phosphatidylcholine hydrogenated (egg) and phosphatidyl choline hydrogenated (soya).Emulsifiers may also be non-ionic surfactants such as poloxamers (forexample Poloxamer 188 and 407), poloxamines, polyoxyethylene stearates,polyoxyethylene sorbitan fatty acid esters or sorbitan fatty acidesters. Ionic surfactants may also be used such as cholic acid anddeoxycholic acid or surface active derivatives or salts thereof. Theemulsifier can also be a mixture of one or more of the aboveingredients. The emulsion may additionally contain other ingredientssuch as buffers, stabilizers and other lipids.

Intralipid® is a fat emulsion for injection. Fat emulsions may containegg yolks, soybean oil, and safflower oil. Intralipid®, marketed in theU.S. as Liposyn II® and Liposyn III® (Abbot Laboratories, Abbott Park,Ill.), may be used as a source of calories and fatty acids to maintainor increase the weight of the patient to whom it is administered, or itmay be used as a vehicle for poorly water-soluble lipophilic drugs thatcannot be injected directly. Intralipid® and Liposyn II® are marketed inboth a 10% and 20% concentration. In accordance with the presentinvention, an emulsion comprising β-lapachone, its derivatives oranalogs, and Intralipid®, or any other pharmaceutically acceptable fatemulsion, may be prepared for parenteral administration to a patient.

Recent in vitro and in vivo studies have shown that β-lapachonedemonstrates significant synergy with other chemotherapeutic andanticancer agents, particularly cis-platinum, and taxane derivatives,such as Taxol® (paclitaxel) (Bristol-Myers Squibb Co., New York, N.Y.).WO00/61142, for example, discloses a method and composition for thetreatment of cancer, which comprises the administration of an effectiveamount of a first compound, a G1 or S phase drug, such as a β-lapachone,in combination with a G2/M drug, such as a taxane derivative. By virtueof both its major functional characteristics—synergy with otherchemotherapy drugs and activity against resistant cells—the use ofβ-lapachone, its derivatives or analogs, may significantly increase therate of long term remission of numerous cancers, including ovarian,breast, prostate, colon, pancreatic, multiple myeloma, malignantmelanoma and non-melanoma skin cancers. β-lapachone, its derivatives oranalogs may also be used to treat proliferation disorders anddermatologic conditions, such as psoriasis.

As recited, the pharmaceutical composition and formulations of thepresent invention are intended for parenteral administration, preferablyintravenous administration. The invention is not, however, limited inthis respect and liquid pharmaceutical compositions and formulations inaccordance with the present invention may be prepared for oralingestion.

Advantageously, pharmaceutical compositions for parenteraladministration comprise a desired amount of β-lapachone, its derivativesor analogs, complexed with HPBCD. Regular β-cyclodextrins are notsuitable for formulations intended for parenteral administration, butmay be used for the preparation of formulations for oral administration.As recited, experimentation has determined that the solubility ofβ-lapachone, its derivatives or analogs, increases linearly with theincrease in HPBCD concentration.

While β-lapachone is the preferred compound for use in the compositionin accordance with the present invention, the invention is not limitedin this respect, and β-lapachone derivatives or analogs, such aslapachol, and pharmaceutical compositions and formulations thereof arepart of the present invention. Such β-lapachone analogs include thoserecited in PCT International Application PCT/US93/07878 (WO 94/04145),which is incorporated by reference herein in its entirety, and whichdiscloses compounds of the formula:

where R and R₁ are each independently hydrogen, substituted andunsubstituted aryl, substituted and unsubstituted alkenyl, substitutedand unsubstituted alkyl and substituted or unsubstituted alkoxy. Thealkyl groups preferably have from 1 to about 15 carbon atoms, morepreferably from 1 to about 10 carbon atoms, still more preferably from 1to about 6 carbon atoms. The term alkyl unless otherwise modified refersto both cyclic and noncyclic groups, although of course cyclic groupswill comprise at least three carbon ring members. Straight or branchedchain noncyclic alkyl groups are generally more preferred than cyclicgroups. Straight chain alkyl groups are generally more preferred thanbranched. The alkenyl groups preferably have from 2 to about 15 carbonatoms, more preferably from 2 to about 10 carbon atoms, still morepreferably from 2 to 6 carbon atoms. Especially preferred alkenyl groupshave 3 carbon atoms (i.e., 1-propenyl or 2-propenyl), with the allylmoiety being particularly preferred. Phenyl and napthyl are generallypreferred aryl groups. Alkoxy groups include those alkoxy groups havingone or more oxygen linkage and preferably have from 1 to 15 carbonatoms, more preferably from 1 to about 6 carbon atoms. The substituted Rand R₁ groups may be substituted at one or more available positions byone or more suitable groups such as, for example, alkyl groups such asalkyl groups having from 1 to 10 carbon atoms or from 1 to 6 carbonatoms, alkenyl groups such as alkenyl groups having from 2 to 10 carbonatoms or 2 to 6 carbon atoms, aryl groups having from six to ten carbonatoms, halogen such as fluoro, chloro and bromo, and N, O and S,including heteroalkyl, e.g., heteroalkyl having one or more hetero atomlinkages (and thus including alkoxy, aminoalkyl and thioalkyl) and from1 to 10 carbon atoms or from 1 to 6 carbon atoms.

Other β-lapachone analogs contemplated in accordance with the presentinvention include those described in U.S. Pat. No. 6,245,807, which isincorporated by reference herein in its entirety, and which disclosesβ-lapachone analogs and derivatives having the stricture:

where R and R₁ are each independently selected from hydrogen, hydroxy,sulfhydryl, halogen, substituted alkyl, unsubstituted alkyl, substitutedalkenyl, unsubstituted alkenyl, substituted aryl, unsubstituted aryl,substituted alkoxy, unsubstituted alkoxy, and salts thereof, where thedotted double bond between the ring carbons represents an optional ringdouble bond.

Additional β-lapachone analogs and derivatives are recited in PCTInternational Application PCT/US00/10169 (WO00/61142), which isincorporated by reference herein in its entirety, and which disclosecompounds of the structure:

where R₅ and R₆ may be independently selected from hydroxy, C₁–C₆ alkyl,C₁–C₆ alkenyl, C₁–C₆ alkoxy, C₁–C₆ alkoxycarbonyl, —(CH₂)_(n)-phenyl;and R₇ is hydrogen, hydroxyl, C₁–C₆ alkyl, C₁–C₆ alkenyl, C₁–C₆ alkoxy,C₁–C₆ alkoxycarbonyl, —(CH₂)_(n)-amino, —(CH₂)_(n)-aryl,—(CH₂)_(n)-heteroaryl, —(CH₂)_(n)-heterocycle, or —(CH₂)_(n)-phenyl,wherein n is an integer from 0 to 10.

Other β-lapachone analogs and derivatives are disclosed in U.S. Pat.Nos. 5,763,625, 5,824,700 and 5,969,163, as well is in scientificjournal articles, such as Sabba et al, J Med Chem 27:990–994 (1984),which discloses β-lapachone with substitutions at one or more of thefollowing positions: 2-, 8- and/or 9-positions. See also Portela et al.,Biochem Pharm 51:275–283 (1996) (substituents at the 2- and9-positions); Maruyama et al, Chem Lett 847–850 (1977); Sun et al.,Tetrahedron Lett 39:8221–8224 (1998); Goncalves et al, Molecular andBiochemical Parasitology 1:167–176 (1998) (substituents at the 2- and3-positions); Gupta et al., Indian Journal of Chemistry 16B: 35–37(1978); Gupta et al., Curr Sci 46:337 (1977) (substituents at the 3- and4-positions); DiChenna et al., J Med Chem 44: 2486–2489 (2001)(monoarylamino derivatives). Each of the above-mentioned references areincorporated by reference herein in their entirety.

More preferably, analogs and derivatives contemplated by the presentapplication are intended to encompass compounds having the generalformula I and II:

where the dotted double bond between the ring carbons represents anoptional ring double bond and where R and R₁ are each independentlyselected from hydrogen, hydroxy, sulfhydryl, halogen, substituted alkyl,unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl,substituted aryl, unsubstituted aryl, substituted alkoxy, unsubstitutedalkoxy, and salts thereof. The alkyl groups preferably have from 1 toabout 15 carbon atoms, more preferably from 1 to about 10 carbon atoms,still more preferably from 1 to about 6 carbon atoms. The term alkylrefers to both cyclic and noncyclic groups. Straight or branched chainnoncyclic alkyl groups are generally more preferred than cyclic groups.Straight chain alkyl groups are generally more preferred than branched.The alkenyl groups preferably have from 2 to about 15 carbon atoms, morepreferably from 2 to about 10 carbon atoms, still more preferably from 2to 6 carbon atoms. Especially preferred alkenyl groups have 3 carbonatoms (i.e., 1-propenyl or 2-propenyl), with the allyl moiety beingparticularly preferred. Phenyl and napthyl are generally preferred arylgroups. Alkoxy groups include those alkoxy groups having one or moreoxygen linkage and preferably have from 1 to 15 carbon atoms, morepreferably from 1 to about 6 carbon atoms. The substituted R and R₁groups may be substituted at one or more available positions by one ormore suitable groups such as, for example, alkyl groups having from 1 to10 carbon atoms or from 1 to 6 carbon atoms, alkenyl groups having from2 to 10 carbon atoms or 2 to 6 carbon atoms, aryl groups having from sixto ten carbon atoms, halogen such as fluoro, chloro and bromo, and N, Oand S, including heteroalkyl, e.g., heteroalkyl having one or morehetero atom linkages (and thus including alkoxy, aminoalkyl andthioalkyl) and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms;and where R₅ and R₆ may be independently selected from hydroxy, C₁–C₆alkyl, C₁–C₆ alkenyl, C₁–C₆ alkoxy, C₁–C₆ alkoxycarbonyl,—(CH₂)_(n)-heteroaryl, —(CH₂)_(n)-heterocycle, or —(CH₂)_(n)-phenyl; andR₇ is hydrogen, hydroxyl, C₁–C₆ alkyl, C₁–C₆ alkenyl, C₁–C₆ alkoxy,C₁–C₆ alkoxycarbonyl, —(CH₂)_(n)-amino, -aryl, —(CH₂)_(n)-heteroaryl,—(CH₂)_(n)-heterocycle, or —(CH₂)_(n)-phenyl, wherein n is an integerfrom 0 to 10.

Preferred analogs and derivatives also contemplated by the inventioninclude compounds of the following general formula III:

where R₁ is (CH₂)_(n)—R₂, where n is an integer from 0–10 and R₂ ishydrogen, an alkyl, an aryl, a heteroaromatic, a heterocyclic, analiphatic, an alkoxy, an allyloxy, a hydroxyl, an amine, a thiol, anamide, or a halogen.

Analogs and derivatives also contemplated by the invention include4-acetoxy-β-lapachone, 4-acetoxy-3-bromo-β-lapachone,4-keto-β-lapachone, 7-hydroxy-β-lapachone, 7-methoxy-β-lapachone,8-hydroxy-β-lapachone, 8-methoxy-β-lapachone, 8-chloro-β-lapachone,9-chloro-β-lapachone, 8-methyl-β-lapachone and8,9-dimethoxy-β-lapachone.

Preferred analogs and derivatives also contemplated by the inventioninclude compounds of the following general formula IV:

where R₁–R₄ are each, independently, selected from the group consistingof H, C₁–C₆ alkyl, C₁–C₆ alkenyl, C₁–C₆ alkoxy, C₁–C₆ alkoxycarbonyl,—(CH₂)_(n)-aryl, —(CH₂)_(n)-heteroaryl, —(CH₂)_(n)-heterocycle, or—(CH₂)_(n)-phenyl; or R₁ and R₂ combined are a single substituentselected from the above group, and R₃ and R₄ combined are a singlesubstituent selected from the above groups, in which case —, —is adouble bond.

Preferred analogs and derivatives also contemplated by this inventioninclude dunnione and 2-ethyl-6-hydroxynaphtho[2,3-b]-furan-4,5-dione.

Preferred analogs and derivatives also contemplated by the inventioninclude compounds of the following general formula V:

where R₁ is selected from H, CH₃, OCH₃ and NO₂.

Preferred compounds of the above generic formulas are illustrated inFIG. 12.

The solubilities of β-lapachone and its analogs in 40% (w/v) HPBCDsolution as compared to water are shown in Tables 3 and 4. To determinesolubilities, test compounds were first dissolved in ethanol to preparestandard solutions with concentrations in the range 2.5–10 mg/ml, thenwere diluted with to 10 μg/ml with water. UV scans were obtained of the10 μg/ml standard solutions, and the wavelength of maximum absorbanceand the absorbance at the maximum absorbance wavelength were determined.For each test compound 50 μl aliquots of water and of 40% HPBCD wereadded to individual Eppendorf tubes containing approximately 1 mg ofcompound each. The tubes were heated in a 30° C. waterbath, vortexed,then centrifuged at 15,000 rpm for 5 min. This step was repeated, thenthe tubes were cooled to room temperature for 1 hour, then werecentrifuged again. The supernatant from each tube was diluted with waterinto the appropriate absorbance range, and the UV absorbance wasmeasured at the compound's absorbance maximum wavelength. Theconcentrations of these saturated solutions were then calculated byratio to the 10 μg/ml ethanolic standard solutions. As shown in Tables 3and 4 aqueous solubilities of the test compounds were increased by 7 to323 fold in the presence of 40% HPBCD.

TABLE 3 Absorbance values of β-lapachone and its analogs in aqueous andin 40% (w/v) hydroxypropyl-β-cyclodextrin (HPBCD) solution WavelengthAbsorbance of at Maximum 10 μg/ml Aqueous Solution HPBCD SolutionAbsorbance, standard Dil Dil Compound (nm) solution Factor AbsorbanceFactor Absorbance β-Lapachone 258 1.041 10 0.452 2000 0.73  (βL)3-Bromo-βL 256 0.788 10 0.286 1000 0.545 3,4-dehydro-βL 262 0.71  100.171 1000 0.41  Dunnione 262 0.944 100  0.449 5000 0.322 4-Acetoxy-βL254 0.89  50 0.506  500 0.373 4-Hydroxy-βL 254 0.973 50 0.742  500 0.5964-keto-βL 280 1.216 50 1.224  500 1.07  3-Hydroxy-βL 256 0.995 200 0.687 2000 0.727 3-(3-methyl-2- 258 0.697 20 0.233 1000 0.284butenyl)-βL

TABLE 4 Solubilities of β-lapachone and its analogs in aqueous and in40% (w/v) hydroxypropyl-β-cyclodextrin (HPBCD) solution SolubilitySolubility Solubility in Water in HPBCD Enhancement Compound (μg/ml)Solution (mg/ml) (−Fold) β-Lapachone (βL) 43 14.0 323 3-Bromo-βL 36 6.9191 3,4-dehydro-βL 24 5.8 240 Dunnione 476 17.0 36 4-Acetoxy-βL 284 2.17.4 4-Hydroxy-βL 381 3.1 8.0 4-keto-βL 503 4.4 8.7 3-Hydroxy-βL 138114.6 10.6 3-(3-methyl-2- 67 4.1 61 butenyl)-βL

Other particular formulations in accordance with the present inventionare set forth herein below and in the Examples section. In general, theβ-lapachone, its derivative and analog, compounds may be prepared in anumber of ways well known to one skilled in the art of organicsynthesis. β-lapachone and its derivatives and analogs may besynthesized using methods generally described below, together withsynthetic methods known in the art of synthetic organic chemistry, orvariations thereon as appreciated by those skilled in the art. Preferredmethods include, but are not limited to, those synthesis and formulationmethods described herein.

Numerous methods are known in the art for synthesizing β-lapachoneand/or derivatives or analogs thereof A first method is described inSchaffner-Sabba, K., et al., β-Lapachone: Synthesis of Derivatives andActivities in Tumor Models, J. Med. Chem., 27, (1984) 990–994, and isknown as the potassium salt method. A second method is described in Sun,J. S. et al., A Preparative Synthesis of Lapachol and RelatedNaphthoquinones, Tetrahedron Letters, 39 (1998) 8221–8224), and is knowas the lithium salt method. These two methods both initially producelapachol, an intermediate from which β-lapachone is synthesized. Both ofthese methods require the formation of a metal salt. Additionally,Amaral, A., et al., in The Total Synthesis of β-lapachone, J.Heterocyclic Chem., 29 (1992) 1457–1460, describes the synthesis ofβ-lapachone α-naphthol in eight steps and results in an overall yield ofonly 23%. In U.S. Pat. No. 5,763,625, lapachol is first converted into3-bromolapachone, which is then converted in a two-step sequence into3-hydroxy-β-lapachone. Furthermore, as described in co-pending U.S.patent application Ser. No. 09/975,776, unlike the reported methods inwhich a metal (lithium or potassium) salt of 2-hydroxy-1,4-naphtoquinonewas prepared in situ by addition of lithium hydride or separately byaddition of potassium hydroxide to the quinone solution as the firststep and then reacting the metal salt with bromide compound to formlapachol, the process described in co-pending U.S. patent applicationSer. No. 09/975,776 eliminates this first step and commences directlywith 2-hydroxy-1,4-naphthoquinone to react with1-bromo-3-methyl-2-butene in the presence of sodium iodide and a weakbase such as triethylamine, pyridine, trimethylamine,N,N-diisopropylethylamine, 2,6-lutidine, to form lapachol from whichβ-lapachone is subsequently synthesized.

As discussed above, β-lapachone as a single agent has been shown to havesignificant cytotoxic activity for a wide variety of cancel cell lines,with IC₅₀ values in the low (1–10) micromolar range. In vitro studieshave demonstrated that these micromolar concentrations of β-lapachonetotally abolished colony formation when applied to tumor cell culturesin combination with IC₅₀ levels of Taxol®. These studies have furthershown that β-lapachone acts synergistically with Taxol®, which containsthe active compound paclitaxel, to significantly augment effectivenessof either agent alone without attendant increases in toxicity (Li, C. J.et al., Proc Natl Acad Sci U.S.A. 96 (1999) 13369).

Potent inhibition of in vivo tumor growth by β-lapachone plus Taxol® hasbeen demonstrated in a xenograft model of human ovarian cancer in nudemice. Potent antitumor activity has also been demonstrated in femalenude mice bearing human breast cancer xenografts (discussed in detail inthe Examples below).

Solubilized β-lapachone, its derivatives and analogs, may also becombined with other taxane derivatives and anticancer agents. In thecombination, solubilized β-lapachone, its derivatives and analogs, maybe admixed with the anticancer agent or taxane derivative, and providedin a single vial, or they may each be provided in a separate vial. Whenthe solubilized β-lapachone, its derivatives and analogs, and theanticancer agent or taxane derivative is provided in separate vials, thecontents of each vial may be administered to the patient simultaneouslyor sequentially.

In another embodiment, solubilized β-lapachone, its derivatives andanalogs, may be administered in combination with radiation therapy.Advantageously, a patient will undergo radiation therapy a predeterminednumber of hours prior to or following β-lapachone, its derivatives andanalogs, administration as determined by the medical clinician treatingthe patient.

The type and amount of β-lapachone, its derivatives and analogs, and theHPBCD or other carrier used will vary widely depending on the species ofthe warm blooded animal or human, body weight, and tumor being treated.Likewise, the dosage administered will vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration, the age, health andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; and the effectdesired.

The dosage administered will vary depending upon known factors such asthe pharmacodynamic characteristics of the particular active ingredient,and its mode and route of administration; age, sex, health, metabolicrate, absorptive efficiency and/or weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment; and the effect desired. In a preferred embodiment, the dosagecan be between approximately 0.1 mg/kg to 10 mg/kg administered frombetween twice weekly to once every four weeks.

As used herein, the term “therapeutically effective amount” means thatamount of a drug or pharmaceutical agent that will elicit the biologicalor medical response of a tissue, system animal or human that is beingsought by a researcher or clinician.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. A dosage unit may comprise a single compound,i.e., β-lapachone, its derivatives and analogs, or mixtures thereof withother compounds or other cancer inhibiting compounds or tumor growthinhibiting compounds or anti-viral compounds. Compositions suitable forparenteral administration advantageously include aqueous sterileinjection solutions, but may also include non-aqueous solutions, whichmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. The formulations may bepresent in unit-dose or multi-dose containers, for example, sealed inampules and vials, and as discussed herein, may be stored in lyophilizedcondition requiring only the addition of the sterile liquid carrier, forexample, water, for injections, immediately prior to use. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsas known in the art for the preparation of such solutions. Thespecifications for the dosage unit forms of the invention are dictatedby and directly dependent on the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

It should be understood that in addition to the ingredients particularlymentioned with regard to the specific compositions and formulations ofthe present invention, the compositions and formulations of thisinvention may include other agents convention in the art having regardto the type of formulation in question, for example, those suitable fororal administration may include flavoring and coloring agents.

In addition to the complex of β-lapachone, its derivatives and analogs,with HPBCD in accordance with the present invention, pharmaceuticalcompositions suitable for parenteral administration via injection orinfusion may also include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), oil and suitable mixtures thereof. In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringeability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin. Parenteral and intravenouscompositions may also include minerals and other materials to facilitatetheir compatibility with the type of injection or delivery system to beused. Additionally, solutions for parenteral administration may containa water soluble salt of the active compound, suitable stabilizingagents, and if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propyl-paraben,and chlorobutanol.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The β-lapachone, derivative or analog complexesdescribed herein can be freeze-dried, then reconstituted in aqueoussolution and be substantially soluble (see Example 5 below).

For oral administration in liquid dosage form, the oral drug componentsare preferably combined with β-cyclodextrin and more preferablyhydroxylpropyl-β-cyclodextrin, however the invention is not limited inthis respect, and the oral drug components may be combined with anyoral, non-toxic, pharmaceutically acceptable inert carriers such asethanol, glycerol, water, oils and the like. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents, andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like. Liquid dosage forms for oraladministration can also contain coloring and flavoring to increasepatient acceptance.

Additional examples of suitable liquid dosage forms may includesolutions or suspensions in water, pharmaceutically acceptable fats andoils, alcohols or other organic solvents, including esters, emulsions,syrups or elixirs, suspensions, solutions and/or suspensionsreconstituted from non-effervescent granules and effervescentpreparations reconstituted from effervescent granules. Such liquiddosage forms may also contain, additional solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, thickeners,and melting agents.

The active compounds may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

The active compounds of this invention are intended for administrationas treatment for cancer and the inhibition of tumors, by any means thatproduces contact of the active compounds with the agent's site of actionin the body. As recited, the preferred mode of administering theβ-lapachone, its derivatives and analogs, active ingredient is viaparenteral administration, preferably intravenous administration (bolusor infusion). The invention is not however limited in this respect, andthe active ingredients in accordance with this invention can beadministered by any conventional means available for use in conjunctionwith pharmaceuticals, either as individual therapeutic agents or in acombination with other therapeutic agents with the intention ofinhibiting tumors. For example, the active compounds may also beadministered intraperitoneally, subcutaneously, or intramuscularly. Theactive compounds may also be formulated for topical administration forthe treatment of skin cancers such as basal-cell carcinoma,squamous-cell cancer, Kaposi's sarcoma and melanoma. The activecompounds can be administered alone, but generally are administered witha pharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The present invention also includes methods for treating cancer byadministering to a patient the compositions and formulations of thepresent invention. In a preferred embodiment, the method comprises theparenteral administration of the compositions and formulations to apatient, preferably via intravenous injection or infusion as describedabove. In another embodiment, the method comprises the topicaladministration of the compositions and formulation of the invention.“Topical application”, “applied topically”, “topical administration” and“administered topically”, are used interchangeably to mean the processof applying or spreading one or more compositions according to theinstant invention onto the surface of the skin of a subject in needthereof. Topical formulations may be comprised of an oil-in-water creamemulsion but is not limited in this respect. Topical formulationscontemplated by the present invention may include delayed releasecompositions capable of producing a slow release of the β-lapachoneanalogs and derivatives.

The formulations for topical administration may optionally contain awide variety of additional components intended to improve the overalldesirability, visual appearance, physical properties and/or physicalfeel, but provided that such optional additives are physically andchemically compatible with the essential components described herein(supra), and do not unduly impair stability, safety or efficacy.Optional additives may be dispersed, dissolved or the like in thecarrier of the present compositions. Optional additives include possibleaesthetic agents, (e.g., absorbents including oil absorbents in the formof cosmetic clays and polymeric absorbents), abrasives, anti-cakingagents, antifoaming agents, additional anti-microbial agents, binders,buffering agents, bulking agents, cosmetic biocides, additionaldenaturants and penetrants (supra), cosmetic astringents, drugastringents, external analgesics, film formers, opacifying agents,fragrances, perfumes, pigments, colorings, skin soothing agents, pHadjusters, chelating agents, UV light absorbing agents, plasticizers,preservatives, preservative enhancers, depiliating agents, desquamationagents and exfoliants, collagens and breakdown products thereof,film-forming agents and the like. Representative examples of suchmaterials are disclosed in Harry's Cosmeticology, 7th Ed., Harry &Wilkinson (Hill Publishers, London 1982); in Pharmaceutical DosageForms—Disperse Systems; Lieberman, Rieger & Banker, Vols. 1 (1988) & 2(1989); Marcel Decker, Inc.; in The Chemistry and Manufacture ofCosmetics, 2nd. Ed., deNavarre (Van Nostrand 1962–1965); and in TheHandbook of Cosmetic Science and Technology, 1st Ed. Knowlton & Pearce(Elsevier 1993).

In other embodiments, compositions for use according to the methods ofthe invention also include compositions having a hydrophilic andhydrophobic phase. Non-limiting examples of suitable non-naturalhydrophobic phase components include: (i) a non-toxic andnon-carcinogenic mixtures of liquid hydrocarbons obtained from petroleum(64, 65); (ii) a non-toxic, non-carcinogenic colloidal system ofnonstraight-chain solid hydrocarbons and high-boiling liquidhydrocarbons in which most of the liquid hydrocarbons are micellar (64,66, 67); (iii) non-toxic and noncarcinogenic straight and branched chainhydrocarbons having from about 7 to about 40 carbon atoms, e.g.,dodecane, isododecane, squalane, cholesterol, hydrogenatedpolyisobutylene, docosane (i.e. a C.sub.22 hydrocarbon), hexadecane,isohexadecane (Permethyl.RTM 101A, Presperse, South Plainfield, N.J.),and the like; (iv) non-toxic and non-carcinogenic C.sub.1–30 alcoholesters of C.sub.1–30 carboxylic acids and of C.sub.2–30 dicarboxylicacids, including straight and branched chain materials as well asaromatic derivatives (e.g., diisopropyl sebacate, diisopropyl adipate,isopropyl myristate, isopropyl palmitate, methyl palmitate, myristylpropionate, 2-ethylhexyl palmitate, isodecyl neopentanoate,di-2-ethylhexyl maleate, cetyl palmitate, myristyl myristate, stearylstearate, isopropyl stearate, methyl stearate, cetyl stearate, behenylbehenrate, dioctyl maleate, dioctyl sebacate, diisopropyl adipate, cetyloctanoate, diisopropyl dilinoleate. (v) non-toxic and non-carcinogenicmono-, di- and triglycerides of C.sub.1–30 carboxylic acids, e.g.,caprilic/capric triglyceride, PEG-6 caprylic/capric triglyceride, PEG-8caprylic/capric triglyceride. (vi) non-toxic and non-carcinogenicalkylene glycol esters of C.sub.1–30 carboxylic acids, e.g., ethyleneglycol mono- and di-esters, and propylene glycol mono- and di-esters ofC.sub.1–30 carboxylic acids e.g., ethylene glycol distearate; (vii)non-toxic and non-carcinogenic propoxylated and ethoxylated derivativesof the foregoing materials; and (viii) non-toxic and non-carcinogenicC.sub.1–30 mono- and poly-esters of monosaccharides andoligosaccharides. Examples of liquid esters that may prove useful in thehydrophobic phase include glucose tetra-oleate; glucose tetra-esters ofsoybean oil fatty acids (unsaturated); mannose tetra-esters of mixedsoybean oil fatty acids; galactose tetra-esters of oleic acid; arabinosetetra-esters of linoleic acid; xylose tetra-linoleate; galactosepenta-oleate; sorbitol tetra-oleate; sorbitol hexa-esters of unsaturatedsoybean oil fatty acids; xylitol penta-oleate; sucrose tetra-oleate;sucrose pentaoletate; sucrose hexa-oleate; sucrose hepta-oleate; sucroseocta-oleate; and mixtures thereof.

In other embodiments, compositions suitable for topical administrationaccording to the present invention include compositions havingalternative carriers. Examples of alternative carriers includecross-linked polymeric compounds containing one or more monomers derivedfrom acrylic acid, substituted acrylic acids, and salts and esters ofthese acrylic acids and the substituted acrylic acids, wherein thecross-linking agent contains two or more carbon-carbon double bonds andis derived from either (i) an acrylic acid homopolymeric polyhydricalcohol, e.g., crosslinked homopolymers of acrylic acid monomer orderivative thereof (e.g., C₁₋₄ alkyl, —CN, or —COOH substituted), wherethe acrylic acid has substituents at the two and three carbon positions(e.g., acrylic acid, methacrylic acid, ethacrylic acid, and mixturesthereof); or (ii) a cross-linked acrylate copolymer having both anacrylic acid monomer (or derivative thereof) and a C₁₋₄ alcohol acrylateester monomer (or derivative thereof), and a second monomer which is along chain alcohol (e.g. C₈₋₄₀) acrylate ester monomer (or derivativethereof), e.g., acrylic acid, methacrylic acid, ethacrylic acid, andmixtures thereof. Combinations of the latter two types of polymers aremay also prove useful in certain compositions for use according to thetreatment methods of the invention.

In addition to treating disorders such as skin cancers, the preparationsof this invention may be used to treat a wide variety of dermatologicconditions or disorders. Dermatologic conditions can be any disorderassociated with the skin. Dermatologic conditions include, but are notlimited to, dermatitis conditions such as: Contact Dermatitis; AtopicDermatitis; Seborrheic Dermatitis; Nummular Dermatitis; ChronicDermatitis of Hands and Feet; Generalized Exfoliative Dermatitis; StasisDermatitis; and Localized Scratch Dermatitis; bacterial infections ofthe skin, such as: Staphylococcal Diseases of the Skin, StaphylococcalScalded Skin Syndrome; Erysipelas; Folliculitis; Furuncles; Carbuncles;Hidradenitis Suppurativa; Paronychial Infections and Erythrasma;superficial fungal infections such as: Dermatophyte Infections; YeastInfections; Candidiasis; and Tinea Versicolor; parasitic infections ofthe skin such as: Scabies; Pediculosis; and Creeping Eruption; disordersof hair follicles and sebaceous; glands such as: Acne; Rosacea; PerioralDermatitis; Hypertrichosis; Alopecia; Pseudofolliculitis Barbae; andKeratinous Cyst; scaling papular diseases, such as: Psoriasis;Pityriasis Rosea; and Lichen Planus; pressure sores; benign tumors andmalignant tumors.

Additional information with regard to the methods of making thecompositions and formulations and the ingredients comprising thecompositions and formulations in accordance with the present inventioncan be found in standard references in the field, such as for example,“Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easter, Pa.,15^(th) Ed. (1975).

The present invention also includes pharmaceutical kits which compriseone or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of an active compound.Such kits may further include, if desired, one or more of variousconventional pharmaceutical kit components, such as, for example,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. In a preferred embodiment, a kit is provided for thetreatment of a mammalian cancer comprising at least one vial containingβ-lapachone, or a derivative or analog thereof. In another preferredembodiment, a kit is provided for the treatment of a mammalian tumorcomprising one or more vials containing a complex of a therapeuticallyeffective amount of β-lapachone, or a derivative or analog thereof, anda pharmaceutically acceptable, water-solubilizing carrier molecule andfurther comprising, within in the same vial or a separate vial, ananticancer agent, particularly a taxane derivative.

Printed instructions, either as inserts or as labels, indicatingquantities of the components to be administered, guidelines foradministration, and/or guidelines for mixing the components, may also beincluded in the kit. In the present disclosure it should be understoodthat the specified materials and conditions are important in practicingthe invention but that unspecified materials and conditions are notexcluded so long as they do not prevent the benefits of the inventionfrom being realized.

The invention is further defined by reference to the following examples,which are not meant to limit the scope of the present invention. It willbe apparent to those skilled in the art that many modifications, both tothe materials and methods, may be practiced without departing from thepurpose and interest of the invention.

EXAMPLES

1. Evaluation of Acceptable Solvent Systems Known to SolubilizeHydrophobic Drug Substances

a. Preparation of β-Lapachone and Hydroxypropyl-β-Cyclodextrin (HPBCD)Solution

Various pharmaceutically acceptable solvent systems known to solubilizehydrophobic drug substances were evaluated with β-lapachone. As shown inTable 5 below, solutions meeting the targeted minimum concentration (10mg/ml) were achieved in several of the solutions evaluated. However,none of these systems could be diluted 5× with sterile saline withoutsignificant precipitation of the β-lapachone from solution. In addition,most of these co-solvents and surfactants have their own toxicity andtolerability issues that need to be managed during high dose drugadministration.

TABLE 5 Undiluted 5X dilution* Solvent System (mg/ml) (mg/ml) Poloxamer(20%) 2.0350 0.0331 Povidone K17 (20%) 1.8250 0.0312 Povidone K12 (20%)1.8600 0.0313 Tween 80 11.1700 1.6550 EtOH (76%) 10.6600 0.1025 PEG 40011.6800 0.1400 Propylene Glycol 8.7800 0.0950 Trappsol (20%) 1.46500.0300 *diluted in 0.9% saline

In light of the above, two different strategies were used to enhanceβ-lapachone solubility in aqueous solution. First, β-lapachone wastreated with metal chelating agents, such as calcium and magnesium, toform soluble complexes; second, β-lapachone was treated with thesolubilizing agents β-cyclodextrin and γ-cyclodextrin to form solubleinclusion complexes. In order to evaluate these four reagents,¹⁴C-labeled β-lapachone in a small volume of ethanol was added toaqueous solutions of the reagents (or to PBS buffer as a control), thenthe relative solubility of β-lapachone in each of the solutions wasmeasured in terms of radioactivity remaining in the supernatant aftercentrifugation.

Specifically, to individual 1.5 ml Eppendorf tubes containing 900 μl ofPBS buffer were added the following: 8 mM CaCl₂ in PBS buffer, 8 mM ofMgCl₂ in PBS buffer, 8 mM β-cyclodextrin in PBS buffer, and 8 mMγ-cyclodextrin in PBS buffer. 10 μl of C¹⁴ labeled β-lapachone (40,000CPM, 0.55 μg) in 75% ethanol was then added to each tube. Aftervortexing and centrifuging at 13,000 rpm for 10 min, 100 μl of thesupernatant solution was counted for radioactivity using a BeckmanScintillation Counter. To the remaining mixture, 0.5 μg (50 μl of 10mg/ml solution) or 600 μg of β-lapachone in 75% ethanol was added. Aftervortexing and centrifuging at 13,000 rpm for 10 min, 100 μl of thesupernatant solution was counted again for radioactivity.

When 0.5 μg of β-lapachone was added, almost 100% of the β-lapachone waspresent in the supernatant for all five aqueous solutions. However, when600 μg of β-lapachone was added, only β-cyclodextrin solution retainedmore than 50% of the β-lapachone in the supernatant. The percentage oflabeled β-lapachone in the supernatant was determined by counting in ascintillation counter. FIG. 1 illustrates the relative solubility ofβ-lapachone in aqueous solutions of various solubilizing agents. In FIG.1, solution 1 consisted of PBS buffer, solution 2 consisted of 8 mMCaCl₂ in PBS buffer, solution 3 consisted of 8 mM MgCl₂ in PBS buffer,solution 4 consisted of 8 mM β-cyclodextrin in PBS buffer, and solution5 consisted of 8 mM γ-cyclodextrin in PBS buffer.

b. Effect of Hydroxypropyl-β-Cyclodextrin (HPBCD) Concentration onβ-lapachone Solubility

Because β-cyclodextrin is suitable for oral, but not for parenteral ortopical use, its analog HPBCD was selected for further study. To examinethe effect of HPBCD concentration on β-lapachone solubility, β-lapachonein small volumes of ethanol was added to eight aqueous solutions withvarying concentrations of HPBCD (0–16 mM or 0–25% (w/w)), then relativesolubility was determined by measuring the percentage of radioactivityremaining in the supernatant after centrifugation. In order to eliminatethe possible effect of ethanol and determine if solubility enhancementcan be maintained after lyophilization, the mixtures were lyophilizedand then re-dissolved into the same volume of water. The percentage ofβ-lapachone in the supernatant of the re-dissolved mixture was measuredto ensure complete resolubilization of the lyophilized material.

Specifically, to individual 1.5 ml Eppendorf tubes, sufficient amountsof water, 50 mM HPCD solution ¹⁴C-labeled β-lapachone solution in 75%ethanol, 10 mg/ml β-lapachone solution in ethanol, and 0.9% NaClsolution were added to prepare 1 ml solutions with componentconcentrations listed in the Table 6.

TABLE 6 Tube # 1 2 3 4 5 6 7 8 9 HPCD, Mm 0 0 1 2 4 6 8 12 16¹⁴C-β-lapachone, 60K 60K 60K 60K 60K 60K 60K 60K 60K CPM β-lapachone,,mM 0 1 1 1 1 1 1 1 1 NaCl, % (w/v) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9

After vortexing and centrifuging at 13,000 rpm for 10 min, 100 μl ofsupernatant from teach tube was counted for radioactivity using aBeckman Scintillation Counter. The rest of the mixtures (900 μl each)were lyophilized and then re-dissolved in 900 μl of water. Aftervortexing and centrifuging at 13,000 rpm for 10 min, 100 μg/ml ofsupernatant from each tube was counted again for radioactivity.

FIG. 2 shows that β-lapachone solubility increases with increased HPBCDconcentration, and that the β-lapachone can be fully resolubilizedfollowing lyophilization.

c. Preparation of β-lapachone and Hydroxypropyl-β-Cyclodextrin (HPBCD)Solution by Heating

A β-lapachone/HPBCD solution was prepared without prior solubilizationof the β-lapachone in ethanol solution. β-lapachone was combined withaqueous solutions of HPBCD in varying concentrations and the mixtureswere heated to 70° C., then allowed to cool to room temperature. Thecooled solutions were filtered (0.22μ), and the amount of thesolubilized β-lapachone was measured by HPLC analysis. The solubility ofβ-lapachone in various aqueous solutions of HPBCD is provided in Table7.

TABLE 7 HPBCD β-lapachone Conc. % (m/M) (mg/ml) 50 (325) 19.7 40 (260)15.8 30 (195) 10.8 20 (130) 7.4 10 (65) 3.1

A maximum concentration of 19.7 mg/ml of β-lapachone was achieved in 50%HPBCD solution (highest concentration tested). The addition of saline orethanol did not significantly enhance the solubility of β-lapachone inHPBCD.

d. HPLC Analysis and UV Measurement of β-lapachone Solution in 75%Ethanol and Aqueous Solution of β-lapachone-HPBCD Complex

5 μg/ml solutions of β-lapachone were prepared for HPLC and UV analysisby diluting either 200 μg/ml ethanolic solutions or 5 mg/ml HPBCDsolutions with water. UV measurements at 258 nm were performed usingroutine procedures with 2% ethanol or 200 μg/ml HPBCD as referencesolutions. For HPLC analysis, 100 μl of the resulting 5 μg/mlβ-lapachone solutions was injected into a C¹⁸ reverse phase analyticalcolumn, and a linear gradient from 25% to 75% B buffer in 10 min at flowrate of 1 ml/min was applied. Peaks were detected by UV absorption at258 nm and quantitated by peak area ratio to external standards.

The λ_(max) for β-lapachone was observed at 258 nm from the UV spectrum.UV measurements of β-lapachone solution at 258 nm showed an extinctioncoefficient of 26620

M⁻¹ cm⁻¹ for both β-lapachone alone and the β-lapachone-HPBCD complex.

FIG. 3 shows a typical HPLC chromatogram of a 5 mg/ml β-lapachonesolution in 20% HPBCD, diluted to 5 μg/ml in water for HPLC analysis.β-lapachone elutes at approximately 5.4 min. Chromatograms showed nodifference in retention times and peak integration areas betweenβ-lapachone alone at 5 μg/ml in water and the comparable 5 μl/mlβ-lapachone-HPBCD complex. These results suggest that the β-lapachone isnot complexed with HPBCD at low concentrations (i.e., 5 μg/ml). Whenincreasing quantities of HPBCD were added to the 5 μg/mlβ-lapachone-HPBCD solution, HPLC analysis showed that a peak eluting atthe void volume of the column (retention time of about 1.2 min) andpresumed to be the β-lapachone-HPBCD complex, increased with size with acorresponding reduction of the β-lapachone peak. However, under theanalytical conditions developed for β-lapachone quantitation, whichrequires dilution to 5 μg/ml, the integration of the peak at ˜5.4 minprovides accurate quantitation of the total β-lapachone in the solution.

e. β-lapachone Stability

The stability of β-lapachone solutions either in 75% ethanol or anaqueous β-lapachone-HPBCD complex form was monitored by HPLC analysis.When stored in the dark at room temperatures, the β-lapachone-HPBCDsolution showed significantly better stability than the ethanolicsolution. The HPBCD solution had no detectable degradation product peaksafter 5 days of storage; and a single degradation product peak at about0.1% at a retention time of 3.28 min after 21 days of storage. Bycomparison, ethanolic solutions stored in the dark showed significantloss of the β-lapachone peak after 5 days of storage. Significantstability enhancement was also observed for the β-lapachone-HPBCDsolution as compared to β-lapachone in 75% ethanol solution when bothwere exposed to light with normal room brightness at room temperature.However, the β-lapachone-HPBCD solution is still appreciably degradedupon exposure to light, with degradation products comprising 3.4% oftotal peak area by day 21 of exposure.

The mechanism of degradation of β-lapachone in alcohol solutions hasbeen shown to involve photoreduction to a relatively stable, semireducedquinone radical (Ci, Xiohong, et al., J. Am. Chem. Soc. 1989: 111,1337–1343). In the above studies, the primary degradation product inethanolic solutions was identified as the reduced (hydroquinone) form ofβ-lapachone through retention time comparisons with the product preparedby reduction of β-lapachone with sodium borohydride. This species, whichelutes at approximately 6.9 min has not been detected in HPBCD solutionsof β-lapachone, which seem to show a different degradative pathway.

2. Lyophilization of the β-lapachone/HPBCD Complex Solution

β-lapachone/HPBCD complex solution samples were prepared in accordancewith the procedure set forth in Example 1a and 1c. The samples weretransferred into a lyophilization container and pre-cooled to 40° C. for2 h. Vacuum was applied to the container for 12–20 hours depending onthe sample(s) (number, size, composition and other properties andcharacteristics of the samples) to provide a freeze-dried product.

The lyophilized sample(s) were reconstituted with 5.9 ml of deionizedwater with agitation to provide β-lapachone at 10 mg/ml. The results ofthe samples tested are shown in Table 8.

TABLE 8 β-lapachone/HPBCD system tested (All at 80 mg/vial) Vol- umeFormulation (ml) Time to Dry/Reconstitute HPBCD @ 40%/β-lapachone @ 8Long (~20 hr)/Long (>10 min)  10 mg/ml (density = 1.125) HPBCD @26%/β-lapachone @ 12 Short (~13 hr)/Short (~10 min) 6.7 mg/ml HPBCD @20%/β-lapachone @ 16 Short (~13 hr)/Short (~5 min)   5 mg/ml HPBCD @10%/β-lapachone @ 32 Long (>20 hr) 2.5 mg/mlBased upon these results, HPBCD@40%/β-lapachone@10 mg/ml accomplishedthe solubility requirement for 10 to 100 times dilution. If the solutionis stable under the storage conditions, it is a suitable parenteralsolution without lyophilization. If lyophilization is preferred,HPBCD@20%/β-lapachone@5 mg/ml was demonstrated to be a good choice forspeedy freeze-drying and relatively fast reconstitution.3. In Vitro Study of β-lapachone Combined with Taxol®

Micromolar concentrations of β-lapachone have been shown to totallyabolish colony formation when applied to tumor cell cultures incombination with IC₅₀ levels of Taxol®. In these studies, exponentiallygrowing cells were seeded at 1,000 cells per well in six-well plates andallowed to attach for 48 h. β-lapachone and/or Taxol®, solubilized inDMSO, were added to the wells. Control wells were treated withequivalent volumes of DMSO. After 4 h cells were rinsed and fresh mediumwas added. Cultures were observed daily for 10–20 days and then werefixed and stained. Colonies of greater than 30 cells were scored assurvivors. As shown in FIG. 4, synergistic inhibition of cancer cellsurvival was seen for a wide spectrum of human carcinoma cells ofdifferent histotypes, including ovarian, breast, prostate, melanoma,lung and pancreatic cell lines. β-lapachone or Taxol® alone were muchless effective in decreasing cancer cell colony formation. The decreasedcell survival was shown to be due to death by the MTT and tryptan blueexclusion assays. DNA laddering formation and annexin staining indicatedthat cell death was due to apoptosis.

Drug-drug interaction of β-lapachone and Taxol® was further evaluated intwo ovarian tumor cell lines, OVCAR-3 and MDAH-2774 using isobologramanalysis. The individual IC₅₀ values for each drug were determined andthen combinations of the two drugs at fixed ratios of their IC₅₀concentrations were applied to the cells. Following a 4-day continuousexposure, cell viability was determined by MTT assay. As illustrated inFIGS. 5, 6 and 7, a pattern of synergistic cell kill was demonstrated bythe combination of these two drugs in these cell lines.

In FIG. 5, when interpreting the combination curves, statisticalcomparisons were made with each test combination and the endpoints (100%β-lapachone and 100% Taxol®). A statistically significant observationrequires that a difference exists between the combination (β-lapachoneand Taxol®) absorbance value and both endpoint values (β-lapachone orTaxol® alone). If the majority of the values (≧3 of 5) are statistically(p<0.05) below the line, then synergy is described. In FIG. 6, the drugcombination is shown to be significantly different (p<0.05) than eitherdrug alone at 3 of the 5 combinations evaluated. In FIG. 7, the drugcombination is shown to be significantly more cytotoxic (p<0.05) thaneither drug alone at 5 of the 5 combinations evaluated.

β-lapachone has also been shown to be active againstcis-platinum-resistant cell lines. The ovarian line A2780DDP is highlycis-platinum (cisplatin) resistant, with an IC₅₀ concentration forcisplatin typically >100 μM. As shown in FIG. 8, β-lapachone as a singleagent is equally cytotoxic to both the highly resistant line and to theparent line from which it is derived (A2780s). In testing β-lapachoneagainst the cisplatin-resistant-cell lines, cells were exposed toβ-lapachone solutions for 4 h. The cells were then rinsed and freshmedium was added. Cultures were observed daily for 10–20 days and thenwere fixed and stained. Colonies of greater than 30 cells were scored assurvivors.

4. In Vivo Studies of β-lapachone Combined with Taxol®

Human ovarian cancer cells (36M2, originally isolated from malignantascites) were inoculated by i.p. injection into athymic female nude mice24 h after irradiation. In this model, metastatic foci formapproximately 1 week after inoculation, and tumor nodules of theperitoneum and malignant ascites develop in 4–5 weeks. Ten days aftertumor inoculation (10×10⁶ cells), treatment regimens were initiated. Thecontrol group was treated with vehicle alone. In each typical treatmentcycle, the β-lapachone alone group was treated with 25–50 mg/kg i.p. ofβ-lapachone in Lipiodol solution and the Taxol® alone group was treated

at 1 mg/kg i.p. (Taxol® formulation diluted in Lipiodol), both followed24 h later by i.p. injection of vehicle. In the combination group, nudemice were treated with β-lapachone at 25–50 mg/kg, followed 24 h laterby Taxol® at 1 mg/kg. All groups were treated for 10 cycles, with a1-day break between each cycle. Mice were sacrificed two weeks laterafter the last treatment cycle (on day 50) to assess antitumor activity.Host toxicity was evaluated by general appearance and body weight.

FIG. 9 shows the representative results for one of three independenttherapeutic experiments, each with 6 mice per group. The decrease intumor number versus control was quite pronounced with β-lapachone alone(˜75%). Mice treated with Taxol® alone showed a slightly smaller effect(˜60%), and both groups showed considerable reduction in the size of thetumor nodules and the amount of ascites. In animals treated withβ-lapachone plus Taxol®, no malignant ascites were seen on thelaparotomy, and the peritoneum was clean except for zero to three tinyfoci per mouse. These foci were counted as tumor nodules although theyappeared to be fibrotic scars. Mice treated with the combination regimenappeared healthy and did not lose body weight throughout the treatmentperiod, and no gross abnormalities in the internal organs were noted inthe autopsy.

A similar study in the human ovarian xenograft model was performedcomparing β-lapachone in HPBCD solution with β-lapachone in Lipiodolsolution. As with the previous study, treatment was initiated ten daysafter IP inoculation of 10×10⁶ 36M2 human ovarian cancer cells intoathymic female nude mice, 8 per group (animals were not irradiated priorto tumor inoculation). The control group was treated with vehicle alone.In each typical treatment cycle, β-lapachone alone groups received 25 or10 mg/kg i.p. of β-lapachone in HPBCD solution and the Taxol® alonegroups was treated at 1 mg/kg i.p. (Taxol formulation diluted inLipiodol), both followed 24 h later by i.p. injection of vehicle. In twocombination groups, mice were treated with β-lapachone in HPBCD ateither 25 or 10 mg/kg, followed 24 h later by Taxol® at 1 mg/kg. A thirdcombination group received β-lapachone in Lipiodol at 25 mg/kg, followed24 h later by Taxol® at 1 mg/kg. All groups were treated for 6 cycles,with a 1-day break between each cycle. Mice were sacrificed on about Day50 (about 4 weeks after last treatment) for assessment of antitumoractivity.

FIG. 10 shows the results of this study. Mice treated with β-lapachonein HPBCD solution showed the same reduction in tumor nodules as micetreated with a comparable level of β-lapachone in Lipiodol solution.

Potent anti-tumor activity was also demonstrated in female nude micebearing human breast cancer xenografts (MCF-7 cell line). Treatment ofmice was initiated after subcutaneous tumor nodules reached ˜0.5 cm indiameter. As shown in FIG. 11, mice receiving six cycles of β-lapachone(50 mg/kg i.p. in Lipiodol solution) and Taxol® (1 mg/kg i.p., 24 hafter β-lapachone dose) showed dramatic reduction of tumor volumecompared to controls. Furthermore, tumors in the treated mice did notincrease in size as of the follow-up. In FIG. 11, the volume ofsubcutaneous tumor xenograft is shown in chart A and the body weight ofthe mice measured for 6 weeks after cessation of treatment is shown inchart B.

5. Study of β-lapachone Formulation in Intralipid®

A 10 mg/ml concentrate of β-lapachone in ethanol was prepared. Theconcentrate was diluted 5×to provide 100-500 μl total. A 2 mg/mlconcentration of β-lapachone was prepared in 10% Intralipid® by dropwiseaddition of the ethanolic solution to the Intralipid® with vortexing. Noimmediate evidence of precipitation or emulsion breaking was observed.

This procedure was repeated wherein the concentrate was diluted 10× toprepare 1 mg/ml β-lapachone in 10% Intralipid®. Ethanol solution wasadded dropwise to the Intralipid® with vortexing. No immediate evidenceof precipitation or emulsion breaking was observed. After 3 days, the 2mg/ml preparation had crystals, and the 1 mg/ml preparation showed nochanges. After 6 days, the 1 mg/ml preparation still showed no changes.

6. Single Agent β-lapachone Analog Inhibition

Several preferred β-lapachone analogs and derivatives in accordance withthe present invention as well as a dunnione analog and4-hexanoyloxy-1,2-naphthoquinone (as stated infra, see illustrations inFIG. 12) were evaluated for their growth inhibitory activity as comparedto β-lapachone (CO-501) in six human cancer cells lines: A2780 andA2780/CP (ovarian); MCF-7 (breast); HT-29 (colon); BxPC-3 (pancreas);and A549 (lung). The human tumor cell lines (purchased from ATCC,Rockville, Md.) were cultured at 37° C. (5% CO₂) in RPMI medium (RPMI;Nova Tech, Grand Island, N.Y.) containing 10% fetal bovine serum (FBS,Nova Tech). Aliquots of cells were seeded into each well of 96-wellmicrotiter plates at a final concentration of 10⁴ cells/well andincubated for 24 h prior to exposure to test compounds. Growthinhibitory activity of DMSO solutions of each compound were measured bythe MTS assay after 4 hours of treatment followed by 24-hour incubationwith drug-free medium. The MTS assay is a colorimetric assay based uponthe ability of viable cells to convert MTS, a novel tetrazolium compoundand electron-coupling reagent, to a colored formazan product that issoluble in cell culture medium. The cell concentration is thendetermined by measuring the absorbance of the formazan product at 490nm. Growth inhibition, expressed as IC₅₀, is calculated relative tovehicle-treated control cells. The results of 3 individual assays, eachof which involved 3 replicates for each dose level, are shown in Table9, below.

As expected, a dose-dependent inhibitory effect was observed forβ-lapachone (CO-501) in all six tumor cell lines. The dunnione analog(CO-506) showed an activity profile similar to CO-501, as was predictedfrom the literature. Of the three β-lapachone analogs and derivatives,CO-504 (4-acetoxy β-lapachone) also was very similar to CO-501 ininhibitory activity; CO-503 (4-hydroxy β-lapachone) was significantlyless active across all cell lines; and CO505 was inactive (no growthinhibition observed at concentrations as high as 20 μM). CO-507, anaphthoquinone derivative, was also inactive.

TABLE 9 Growth inhibitory activity of β-lapachone (CO-501) and fiveanalogs and derivatives Growth inhibitory activity (50% effectiveconcentration, IC₅₀, expressed as μM) Cell Line CO-501 CO-503 CO-504CO-505 CO-506 CO-507 Ovarian A2780 (sensitive) Exp 1 2.11 2.35 1.83 *2.47 * Exp 2 3.99 5.29 1.60 * 4.79 * Exp 3 0.876 4.857 1.19 * 3.23 *Mean ± SE 2.32 ± 0.91 4.16 ± 0.92 1.54 ± 0.19 * 3.50 ± 0.68 * OvarianA2780CP (resistant) Exp 1 4.47 7.42 0.678 * 2.73 * Exp 2 1.61 9.384.00 * 3.85 * Exp 3 1.76 14.8 2.56 * 1.53 * Mean ± SE 2.61 ± 0.93 10.5 ±2.2  2.41 ± 0.96 * 2.70 ± 0.67 * Breast MCF-7 Exp 1 2.37 18.8 2.59 *2.64 * Exp 2 4.75 19.4 4.78 * 3.99 * Exp 3 2.10 19.3 4.28 * 3.53 * Mean± SE 3.07 ± 0.84 19.2 ± 0.17 3.88 ± 0.67 * 3.39 ± 0.40 * Colon HT-29 Exp1 5.37 13.2 14.1 * 17.3 * Exp 2 11.4 11.0 6.67 * 13.4 * Exp 3 9.89 12.13.98 * 11.2 * Mean ± SE 8.89 ± 1.82 12.1 ± 0.63 8.25 ± 3.03 * 14.0 ±1.78 * Pancreas BxPC-3 Exp 1 11.6 * 10.5 * 5.69 * Exp 2 5.68 * 13.5 *8.40 * Exp 3 16.5 * 2.32 * 9.22 * Mean ± SE 11.3 ± 3.12 * 8.74 ± 3.33 *7.77 ± 1.07 * Lung A549 Exp 1 4.48 14.3 19.3 * 7.50 * Exp 2 4.73 7.636.87 * 12.7 * Exp 3 18.7 9.77 3.75 * 15.3 * Mean ± SE 9.30 ± 4.69 10.6 ±1.98 9.97 ± 4.74 * 11.8 ± 2.28 * *No growth inhibition at up to 20 μM7. In vivo Testing of β-lapachone in Bg-Nu-Xid Mice

Chemicals. β-lapachone was synthesized and dissolved in 40%hydroxypropyl β-cyclodextrin at a concentration of 10 mg/mL and kept atroom temperature in a dark container. Hydroxypropyl β-cyclodextrin wasdissolved in distilled water at a concentration of 10 mg/mL and kept atroom temperature. MATRIGEL®, a basement membrane matrix was purchasedfrom Becton Dickinson Labware (BD Biosciences, Two Oak Park Drive,Bedford, Mass.) and was dissolved in Dulbecco's modified Eagle's mediumwith 50 μg/mL Gentamycin and kept frozen at −20° C. MATRIGEL® isextracted from the Engelbreth-Holm-Swarm mouse tumor, a tumor rich inextracellular matrix proteins. The major matrix components are laminin,collagen IV, entactin, and heparan sulfate proteoglycan (perlecan); thematrix also contains growth factors, matrix metalloproteinases (MMPs[collagenases]), and plasminogen activators, without any inhibitors ofmetalloproteinases (TIMPs). The MATRIGEL matrix is a solution at 4° C.and gels at room temperature to form a three-dimensional reconstitutedbasement membrane. This model system closely mimics the structure,composition, physical properties, and functional characteristics of thebasement membrane in vivo and provides a physiologically relevantenvironment for studies of cell morphology, biochemical function,migration or invasion, and gene expression. The frozen MATRIGEL® matrixwas thawed overnight at 4° C. before use.

Cell Cultures. RPMI8226 cells (a human multiple myeloma cell line) wereprovided Dr. William Dalton (Lee Moffit Cancer Center, Tampa, Fla.).They were maintained by frequent passages in RPMI 1640 (Cellgro®,Mediatech Inc., Herndon, Va.) containing 10% Fetal Bovine serum (FBS)(GibcoBRL, Life Technologies, Grand Island, N.Y.) supplemented with2×10⁻³M L-Glutamine, 100 units/mL penicillin (Pen), and 100 μg/mLstreptomycin (Cellgro®D, Mediatech Inc., Herndon, Va.) in 162 cm² cellculture flasks (Costar®, Corning Incorporated, Corning, N.Y.). Theexponentially growing cell lines were CD138+, CD38+/CD45RA−, EBVnegative, and pathogen free.

Mice. Forty male 6 week old Bg-Nu-Xid mice (deficient in T, B, and NKcells) were obtained from the FCRDC, Frederick, Bethesda, Md. and housedat the Redstone animal facility at DFCI. These mice have 3 separatemutations—Beige (Bg) autosomal recessive mutation associated withimpaired chemotaxis and motility of macrophages & deficiency of NKcells; the nude (nu) autosomal recessive mutation associated withdepletion of T cells due to thymic agenesis; and the X-linked immunedefect (xid) which produces functional defects of B lymphocytes. Theanimals were raised in a barrier facility in cages with saw dust beddingand laminar air at 19–22° C. Rodent food and sterile drinking water weresupplied ad libitum. The mice were quarantined to rule out developmentof any disease. One mouse died during transport, and 5 others were lostbecause of dehydration (n=2), probable infection (n=2) and excessivebleeding due to trauma (n=1). After 1 week, enrofloxacin (a quinoloneantibiotic) was added in drinking water of all mice. All proceduresinvolving animals were approved by and performed according to guidelinesof the Institutional Animal Care and Use Committee of the Dana FarberCancer Institute (DFCI).

Histologic Analysis. The mice were sacrificed when the tumor reached 20mm in their largest diameter or they became moribund, as per the policyof the Animal Protocol Committee at Dana Farber Cancer Institute. Themice were anesthetized with isoflurane, and retroorbital blood wascollected. The mice were sacrificed by cervical dislocation. The tumorswere dissected from the soft tissue (fascia, muscle, skin, etc.) andwere fixed in 10% neutralized formalin. Liver, spleen, kidneys, lung,heart and brain from each group were also removed and fixed in formalin.The tissues were dehydrated and embedded in paraffin blocks. They weresectioned into slices 5 μm thick, stained with hematoxylin and eosin(HE), and examined by light microscopy for evidence of apoptosis.

Statistical Analysis. Statistical analysis was done using the student's‘t’ test for comparing the differences in tumor volumes and degree ofapoptosis between lapachone and control groups. p value of <0.05 wasconsidered significant.

Design Thirty four mice were included in the study. RPMI 8226 (3×10⁷)multiple myeloma cells were washed 3 times, re-suspended in 100 μL RPMI1640, and injected subcutaneously in the right flank of all mice alongwith 100 μL of MATRIGEL® matrix using a hypodermic 27G needle and 1 mLsyringe. The mice were observed for well being and development of tumorsdaily, and were weighed weekly.

Localized palpable tumors developed in all mice (n=34) by a mean of 7days after injection of RPMI 8226 cells. Once the tumors were palpable,they were measured by hand held vernier calipers in 2 orthogonaldiameters every other day. Thirty-one mice were randomized toβ-lapachone (n=16) and control (n=15) groups. The mice in the controlgroup received 50 mg/kg body weight of 40% hydroxypropyl β-cyclodextrinsolution intraperitoneally at the lower left abdominal area every otherday. The mice in the β-lapachone group received β-lapachone in 40%hydroxypropyl β-cyclodextrin at 50 mg/kg body weight intraperitoneallyevery alternate day (FIG. 13). The usual volume at each injection was125 μL. The diameters of the tumors were recorded, and the volumes werecalculated using standard formula for cylindrical objects i.e.0.523×(Smaller diameter)²×Larger diameter. Mice were sacrificed when thetumor was >20 mm in largest diameter or they became moribund.

This xenograft mouse model is attractive because it is easilyestablished, allows monitoring growth of subcutaneous tumors by externalmeasurements, and can be used to study the effects of variouschemotherapeutic agents. Rapidly growing tumors may show some areas ofapoptosis/necrosis, but in our experience that has not been a majorobstacle in the evaluation of cytotoxicity of various novel potentialtherapeutic agents. Similar models have been reported previously usinganti-gp130 agonist monoclonal antibodies (B1+I2) to study growth andimmortalization of multiple myeloma patient cells (Reme et al. Br JHaematol 114:406, 2001) and using anti-human IL-6R antibody PM1 and antihuman IL-6 antibody MH166, to inhibit growth of IL-6 dependent cell line(S6B45) (Suzuki et al. Eur J Immunol 22:1989, 1992). Various animalmodels for testing multiple myeloma therapeutic agents have beenreported previously (Gado et al. Haematologica 86: 227, 2001; Dallas etal. Blood 93:1697, 1999; Manning et al. Immunol Cell Biol 73:326, 1995;Takura et al. Cancer Res 26:2564, 1996; Potter et al. J Exp Med 161:996,1985; Yaccoby et al. Blood 92:2908, 1998; Urashima et al. Blood 90:754,1997).

Determination of β-lapachone and cyclodextrin toxicity. Mice in bothgroups tolerated β-lapachone and hydroxypropyl β-cyclodextrin well. Nomice died in either group and all gained weight (FIG. 14). There was noevidence of overt toxicity of β-lapachone or hydroxypropylβ-cyclodextrin in either cohort. One mouse developed iatrogenicintra-peritoneal hemorrhage after injection of β-lapachone whichresolved in 36 h. Mild tubular vacuolization of kidneys in bothβ-lapachone and control groups was also found. Since this effect waspresent in both groups, hydroxypropyl β-cyclodextrin is implicated, ashas been previously reported for cyclodextrins (Frank et al. Am J Pathol83:367, 1976) and these nephrotoxic changes are reversible withcessation of treatment (Donaubauer et al. Regul Toxicol Pharmacol27:189, 1998).

Effects on β-lapachone tumor volume. Mice in control group received amaximum of 6 doses of hydroxypropyl β-cyclodextrin, whereas the mice inthe β-lapachone group were able to receive a maximum of 8 doses of thisagent. There was a statistically significant decrease in tumor volume ofmice in β-lapachone group (p=0.007) versus control group (FIG. 15) byday 11. Importantly, survival was greater at day 5, 7, 9, 11, 13, 15 and17 in the β-lapachone group compared to controls (FIG. 16), suggestiveof slower tumor growth in β-lapachone group.

Histologic Staining. Histopathologic examination revealed that tumorswere not encapsulated and were locally invasive to soft tissues,including muscle, without any distant metastasis. Tumors werevascularized by blood vessels of murine origin, with a minor variabledegree (0–10%) of cell death primarily in their cores. Apoptosis wasassessed histopathologically on the basis of (1) chromatin condensationand aggregation near the nuclear membrane with convolution of thenuclear membrane; (2) enlarged and abnormally granular nucleolus; (3)shrinkage and rounding of cells; (4) blebbing of cell membranes; and (5)minor dilation of endoplasmic reticulum and mitochondria There was astatistically significant increase in MM cell apoptosis (p=0.001) intumors in the β-lapachone (mean±SD=41.1%±12.7) versus control(mean±SD=20.0%±10.4) groups, as assessed by two blinded independentobservers using light microscopy (FIG. 17A).

There was no microscopic evidence of any toxicity of β-lapachone orhydroxypropyl β-cyclodextrin on liver, heart, lung, brain, and spleen inmice in either β-lapachone or control groups (FIG. 17B, C, D, E, F).Kidneys from mice in both groups showed mild tubular vacuolization,suggesting tubular toxicity of hydroxypropyl β-cyclodextrin (FIG. 17G,H). This toxicity is not expected to be seen at the anticipatedtreatment doses in humans based on with previous drugs formulated inHPBCD. These results indicate that β-lapachone, formulated in HPBCD, issafe and effective at inhibiting tumor cell growth, associated withprolonged host survival in vivo. Thus it can be concluded that beta laphas significant anti-tumor activity with minimal toxicity and can beused to treat multiple myeloma in vivo.

EQUIVALENTS

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims, which follow. In particular, it is contemplated by theinventors that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The claims presented arerepresentative of the inventions disclosed herein. Other, unclaimedinventions are also contemplated. It is to be understood that thedrawings are not necessarily drawn to scale, but that they are merelyconceptual in nature. Applicants reserve the right to pursue suchinventions in later claims.

1. A method for treating cancer comprising administering to a patient apharmaceutical composition comprising a therapeutically effective amountof Beta-lapachone, or a derivative or analog thereof, and apharmaceutically acceptable solubilizing carrier molecule, wherein saidcancer is selected from the group consisting of multiple myeloma, ovary,breast, melanoma, colon, pancreas, lung and prostate and wherein saidsolubilizing carrier molecule is a beta-cyclodextrin.
 2. The method ofclaim 1, wherein the pharmaceutical composition when diluted with anaqueous solution for parenteral administration, remains soluble in theaqueous solution.
 3. The method of claim 2, wherein the pharmaceuticalcomposition comprises a complex or solution of the therapeuticallyeffective amount of Beta-lapachone, or a derivative or analog thereof,and the pharmaceutically acceptable solubilizing carrier molecule.
 4. Amethod for treating cancer comprising administering to a patient aformulation of Beta-lapachone, or a derivative or analog thereof, and apharmaceutically acceptable solubilizing carrier molecule, wherein thecomplex can be freeze-dried and when subsequently reconstituted inaqueous solution is soluble, wherein said cancer is selected from thegroup consisting of multiple myeloma, ovary, breast, melanoma, colon,pancreas, lung and prostate and wherein said solubilizing carriermolecule is a beta-cyclodextrin.
 5. The method of claim 4, wherein theformulation comprises a complex or solution of the Beta-lapachone, or aderivative or analog thereof, and the pharmaceutically acceptablesolubilizing carrier molecule.
 6. The method of claim 4, wherein theformulation is administered parenterally.
 7. The method of claim 6,wherein said formulation comprises a dosage unit in the range between0.1 mg/kg to 10 mg/kg administered from between twice weekly to onceevery four weeks.
 8. A method for treating cancer comprisingadministering to a patient a pharmaceutical composition comprising atherapeutically effective amount of Beta-lapachone, or a derivative oranalog thereof and a pharmaceutically acceptable solubilizing carriermolecule, and further comprising a second anticancer agent and apharmaceutically acceptable carrier, wherein said cancer is selectedfrom the group consisting of multiple myeloma, ovary, breast, melanoma,colon, pancreas, lung and prostate and wherein said solubilizing carriermolecule is a beta-cyclodextrin.
 9. The method of claim 8, wherein thepharmaceutical composition comprises a complex or solution of thetherapeutically effective amount of Beta-lapachone, or a derivative oranalog thereof, and the pharmaceutically acceptable solubilizing carriermolecule, and further comprising the second anticancer agent and apharmaceutically acceptable carrier.
 10. The method of claim 8, whereinthe second anticancer agent is a taxane derivative.
 11. The method ofclaim 10, wherein the taxane derivative is paclitaxel.
 12. The method ofclaim 8, wherein the pharmaceutical composition is administeredparenterally.
 13. The method of claim 8, wherein the pharmaceuticalcomposition when diluted with an aqueous solution for parenteraladministration, remains soluble in the aqueous solution.
 14. The methodof claim 13, wherein the pharmaceutical composition comprises a complexor solution of the therapeutically effective amount of Beta-lapachone,or a derivative or analog thereof and the pharmaceutically acceptablesolubilizing carrier molecule, which when diluted with the aqueoussolution for parenteral administration, remains soluble in the aqueoussolution.
 15. A method for treating cancer comprising administering to apatient a formulation of Beta-lapachone, or a derivative or analogthereof, and a pharmaceutically acceptable solubilizing carriermolecule, wherein the formulation can be freeze-dried and whensubsequently reconstituted in aqueous solution is soluble and furthercomprising a second anticancer agent and a pharmaceutically acceptablecarrier, wherein said cancer is selected from the group consisting ofmultiple myeloma, ovary, breast, melanoma, colon, pancreas, lung andprostate and wherein said solubilizing carrier molecule is abeta-cyclodextrin.
 16. The method of claim 15, wherein the formulationcomprises a complex or solution of the Beta-lapachone, or a derivativeor analog thereof, and the pharmaceutically acceptable solubilizingcarrier molecule, and further comprising the second anticancer agent anda pharmaceutically acceptable carrier.
 17. A method for treating cancercomprising first administering to a patient a pharmaceutical compositioncomprising a therapeutically effective amount of Beta-lapachone, or aderivative or analog thereof, and a pharmaceutically acceptablesolubilizing carrier molecule, and subsequently subjecting said patientto radiation therapy, wherein said cancer is selected from the groupconsisting of multiple myeloma, ovary, breast, melanoma, colon,pancreas, lung and prostate and wherein said solubilizing carriermolecule is a beta-cyclodextrin.
 18. A method for treating skin canceror a dermatologic condition comprising administering to a patient atherapeutically effective amount of a pharmaceutical compositioncomprising a therapeutically effective amount of a Beta-Lapachone, or aderivative or analog thereof, and a pharmaceutically acceptablesolubilizing carrier molecule, wherein said solubilizing carriermolecule is a beta-cyclodextrin.
 19. The method of claim 18, wherein thepharmaceutical composition is administered topically.
 20. The method ofclaim 1, wherein the pharmaceutical composition is administeredparenterally.
 21. The method of claim 1, wherein the pharmaceuticalcomposition comprises a complex or solution of the therapeuticallyeffective amount of Beta-lapachone, or a derivative or analog thereof,and the pharmaceutically acceptable solubilizing carrier molecule, whichwhen diluted with the aqueous solution for parenteral administration,remains soluble in the aqueous solution.
 22. The method of claim 17,wherein the pharmaceutical composition when diluted with an aqueoussolution for parenteral administration, remains soluble in the aqueoussolution.
 23. The method of claim 17, wherein the pharmaceuticalcomposition comprises a complex or solution of the therapeuticallyeffective amount of Beta-lapachone, or a derivative or analog thereof,and the pharmaceutically acceptable solubilizing carrier molecule, andfurther comprising the second anticancer agent and a pharmaceuticallyacceptable carrier.
 24. The method of claim 18, wherein thepharmaceutical composition comprises a complex or solution of thetherapeutically effective amount of Beta-lapachone, or a derivative oranalog thereof, and the pharmaceutically acceptable solubilizing carriermolecule, and further comprising the second anticancer agent and apharmaceutically acceptable carrier.
 25. The method of claim 15, whereinthe pharmaceutical composition is administered parenterally.
 26. Themethod of claim 17, wherein the pharmaceutical composition isadministered parenterally.
 27. The method of claim 17, wherein thepharmaceutical composition comprises a complex or solution of thetherapeutically effective amount of Beta-lapachone, or a derivative oranalog thereof and the pharmaceutically acceptable solubilizing carriermolecule, which when diluted with the aqueous solution for parenteraladministration, remains soluble in the aqueous solution.
 28. The methodof claim 1, wherein said pharmaceutical composition comprises a dosageunit in the range between 0.1 mg/kg to 10 mg/kg administered frombetween twice weekly to once every four weeks.
 29. The method of claim8, wherein said pharmaceutical composition comprises a dosage unit inthe range between 0.1 mg/kg to 10 mg/kg administered from between twiceweekly to once every four weeks.
 30. The method of claim 15, whereinsaid formulation comprises a dosage unit in the range between 0.1 mg/kgto 10 mg/kg administered from between twice weekly to once every fourweeks.
 31. The method of claim 17, wherein said pharmaceuticalcomposition comprises a dosage unit in the range between 0.1 mg/kg to 10mg/kg administered from between twice weekly to once every four weeks.32. The method of claim 18, wherein said pharmaceutical compositioncomprises a dosage unit in the range between 0.1 mg/kg to 10 mg/kgadministered from between twice weekly to once every four weeks.
 33. Themethod of claim 15, wherein the second anticancer agent is a taxanederivative.
 34. The method of claim 33, wherein the taxane derivative ispaclitaxel.